CN1037925A - Monoclonal antibody - Google Patents
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- CN1037925A CN1037925A CN89102525A CN89102525A CN1037925A CN 1037925 A CN1037925 A CN 1037925A CN 89102525 A CN89102525 A CN 89102525A CN 89102525 A CN89102525 A CN 89102525A CN 1037925 A CN1037925 A CN 1037925A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57423—Specifically defined cancers of lung
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
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- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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- G01N33/57469—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving tumor associated glycolinkage, i.e. TAG
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/77—Internalization into the cell
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Abstract
The present invention relates to a kind of new tumor associated antigen, this antigen be a kind of molecular weight 110,000-140, the cell surface glycoprotein in 000 dalton's scope is present in the various cancers that comprise squamous cell cancer and gland cancer.The present invention also comprise can with the hybridoma cell line of this antigen reactive antibody, generation antibody of the present invention and the method for in the diagnosis of cancer and treatment, using this antibody.
Description
The present invention relates to a kind of new tumor associated antigen, this antigen is a kind of cell-surface antigens that is present in the various cancers that comprise squamous cell cancer and gland cancer.The present invention also comprise can with this antigen reactive antibody, can produce the hybridoma cell line of antibody of the present invention and use these antibody diagnosis and the treatment method for cancer.
The early diagnosis of malignant tumour and the evaluation of tumor type are most important to the clinical treatment of tumour.The present invention includes can with the antigen reactive monoclonal antibody expressed on the human cancer cell.Though about having narration (United States Patent (USP) 4,708,930 with the MONOCLONAL ANTIBODIES SPECIFIC FOR method of cancer reaction; European patent bulletin 157,613; Mazauric et al., Cancer Research, 42: 150,1982; Brenner et al., Cancer Research, 42: 3187,1982; Mulshine et al., The Journal of Immunology, 13161: 497,1983; Masuko et al., Japanese Journal for Cancer Research, 76(5): 386,1985), but Shang Weiyou about can with the narration of the antigen reactive monoclonal antibody of the present invention.The squamous cell cancer of lung, head and neck, and the gland cancer of colon and lung are all expressed this antigen, thus can both with antibody response of the present invention.The antigenic extensive distribution of L/1C2 on people's cancer illustrates that the present invention has important diagnostic and treatment is used.
The present invention relates to the relevant glycoprotein antigen of a kind of and tumour of substantially pure form, described antigenic molecular weight is measured as 110 with SDS-PAGE under reductive condition, and 000-140 is in the 000 daltonian scope; This antigen is present in the people's squamous epithelium cancer cell surface by the epithelial cell generation of head, neck and lung; This antigen can with the antibody generation immunoprecipitation that produces by hybridoma cell line L/1C2.
Be that internalization (interalized) takes place after L/1C2 antigen and the antibodies of the present invention unexpectedly.The antigenic internalization of L/1C2 is an importance of treatment plan of the present invention, discharge because this internalization effect causes in the cell of the molten cancer agent that has been connected with antibody of the present invention, this release will antibody combine with L/1C2 antigen form immune complex and subsequently by endocytosis after just can take place.
The invention still further relates to generation can with the hybridoma cell line of the antibody of the above-mentioned glycoprotein antigen relevant reaction with tumour.
The invention still further relates to can with the antibody of the above-mentioned glycoprotein antigen relevant reaction with tumour.
The invention still further relates to the method that L/1C2 antigen in the test sample exists, this method comprises, in this sample, add can with the antibody of above-mentioned tumor associated antigen reaction, and measure the reactive behavior of this antibody and sample.
Antibody of the present invention is particularly useful for discharging cytotoxic agents to expressing the antigenic tumour of L/1C2 in vivo.Render a service greatly after cytotoxic agents links to each other with antibody of the present invention and strengthen.
Fig. 1: the SDS-PAGE (SDS-PAGE) of L/1C2 antigen and L/1C2 monoclonal antibody is analyzed.Each sample carries out electrophoresis by polyacrylamide matrix in the presence of sodium lauryl sulphate.The albumen of migration distance and known molecular amount is compared to determine each subunit of antibody and antigenic molecular weight.Under reductive condition, carry out SDS-PAGE so that each subunit dissociation that disulfide linkage connects.Figure A: under reductive condition, carry out electrophoresis and, show L/1C2 antibody through proteic wall scroll heavy chain of A and wall scroll light chain purifying through the 7-15% of Coomassie blue stain gradient gel.Figure B: immunoprecipitation
3The L/1C2 antigen of H-glycosamine mark with the molecular weight standard thing at the radioautogram after analyzing on the 7-15% gel under the reductive condition.The molecular weight of shown digitized representation standard substance takes advantage of 10
-3Myelomatosis IgG3 negative control not immunity is settled out appraisable antigen.
The cell sorter analysis of Fig. 2: L/1C2 and the reactive behavior of selected tumor cell line.As shown in the figure, USCLS-1, M21 and T222 target cell are suspended with L/1C2 or myelomatosis IgG3 contrast immunoglobulin (Ig) (m-IgG3) and be incubated.Detection reagent is fluorescein-labeled sheep F(ab ')
2Anti-mouse IgG and IgM.Marked average channel fluorescence.
Fig. 3: the internalization kinetics after disclosing L/1C2 antibody and L/1C2 antigen on the T222 cell combines by ultraviolet microscopy.The details of this analysis is seen example 10.Figure A shows 0 o'clock film distinctive bright ring sample fluorescence that dyes.Analysis when figure B is 105 minutes, the painted fluorescence beam of showed cell periphery.Analysis when figure C is 135 minutes shows unique cell inner dyeing, shows the internalization of L/1C2 antibody.
Fig. 4: show that example 12 described bare mouse different species move the growth curve of T222 heterograft in the value model.Figure A shows that L/1C2-DAVLBHYD suppresses the effectiveness of T222 tumor growth.Figure B is a control group, and wherein DAVLBHYD does not combine with cancer cell bonded immunoglobulin (Ig) with a kind of.Figure C is free drug contrast, introduce this contrast and be for can with the activity of DAVLBHYD with can and can not make comparisons with the antigen reactive figure A of L/1C2 immune conjugate with the antigen reactive figure B of L/1C2 immune conjugate.
Use by biosynthesizing
3H-glycosamine labelling human cancerous cell line then can utilize immuno-precipitation to isolate the antigen that is called L/1C2 from the extract of these clones.Through measuring on reductibility SDS-PAGE gradient gel, L/1C2 antigen is the glycoprotein of a kind of molecular weight in 110,000 to 140,000 dalton's scopes.
Hybridoma cell line of the present invention can prepare with the following method: come activate immunity to learn with the clone in a kind of people's cancer source as immunogenic substance and go up relevant splenocyte, merge by splenocyte and murine myeloma cell then, make splenocyte keep survival.Select then and screen produce by this fusion those can with the antigen reactive hybrid cell of L/1C2 (being called hybridoma) or the hybridoma cell line that are present on the various human cancerous cell line (comprising the clone that following table 1 is listed).
Table 1
The representativeness that L/1C2 antigen is fastened at human cancer cell distributes
Clone source/brief comment film fluorescence
Squamous cell cancer
The FADU pharynx+
ME180 uterine cervix (epiderm-like)+
The T222 lung+
The USCLS-1 lung+
5637 bladders+
Transitional cell carcinoma
The T24 bladder+
RT4 bladder (papilloma)+
The J82 bladder+/-
The TCCSUP bladder+
Gland cancer
The WiDr colon+
The HT29 colon+
SK-CO-1 colon (ascites)+
The UCLA/P3 lung+
The DU145 prostate gland+
The PC3 prostate gland+/-
Melanoma
M21 skin-
M14 skin-
Non-transformed cell system
FLOW 2000 tire lung fibroblasts-
Detroit 551 skin flbroblast-
In the table 1, significantly film fluorescence is represented with (+); Hypofluorescence usefulness (+/-) expression; Negative reaction is active in (-) expression.All mensuration are all carried out with respect to the myeloma protein negative control.Target cell system comprises the following clone that derives from American type culture collection (Rockville, MD 20852 ATCC): FaDu(ATCC#HTB43), 5637(ATCC #HTB9), ME180(ATCC #HTB33), SK-CO-1(ATCC #HTB39), PC3(ATCC #CRL1435), DU145(ATCC #HTB81), T24(ATCC #HTB4), RT4(ATCC #HTB2), HT29(ATCC #HTB38), J82(ATCC #HTB1), TCCSUP(ATCC #HTB5) and WiDr(ATCC #CCL218).Other clone comprises: T222(Masui, et al., Cancer:44(3): 1002-7 1984), M14(Chee et al., Cancer Research 36(4): 1503-1509,1976), UCLA/P3(Varki et al., Cancer Research 44:681-687,1984) and M21(Morton et al., Surgery 64(1): 233-240,1968).Flow 2000 derives from Flow Laboratory, Inc., 7655 Old Springhouse Road, Mclean, VA, 22101.
Table 1 has shown that fully L/1C2 antigen is in the extensive distribution that derives from the clone of epithelial tumor.With an Epics-Coulter
Mark IV
(Coulter Electronics, Hialeh FL), estimate the reactive behavior of antibody of the present invention and clone to cytoanalyze by producer's method.The flow cytometry analysis the results are shown in accompanying drawing 2.Pathology sample and healthy tissues to the Freshman tumour are estimated so that diagnostic method of the present invention to be described.The method of measuring the antigenic healthy tissues distribution of L/1C2 with immunoperoxidase (a kind of antibody-enzyme conjugates) method is described in example 6.
Table 2 has illustrated the extensive distribution of L/1C2 antigen on the cancer of Different Organs.
Table 2
With illustrative antibody L/1C2 to people's tumour
Tissue carries out immunoperoxidase staining
Tumor response number/test number
Lung squamous cell cancer 15/15
Head and neck squamous cell cancer 12/12
Adenocarcinoma of colon 17/17
Adenocarcinoma of lung 7/7
Mammary cancer 9/14
Ovarian cancer 6/8
Prostate cancer 4/5
Lymphoma 0/1
Melanoma 0/2
The evaluation that people's healthy tissues is done shows that the L/1C2 antigen presentation is on blood vessel, conduit and the tubule of the some organs that comprise kidney and liver.It also is expressed on intestines and the bronchial epithelial surface.Contain at the same time in the colon sample of tumour and healthy tissues, tumor tissues is darker than normal colonic tissue's dyeing.
Hybridoma technology by Kohler and Milstein narration (Nature, 256:495-497,1975), can utilize this technology to prepare some hybridoma cell line the earliest, makes its secretory product monoclonal anti physical efficiency and L/1C2 antigen-reactive.A kind of general method of preparation these hybridoma cell lines of the present invention is described in example 1, and this method relates to the structure of hybridoma cell line L/1C2, and this clone can obtain from ATCC, and registration number is ATCC #B9682.This area professional will know, this ⒚ sole ò pulls out admittedly to carry on the back to chant and melts squid Fu Ke/1C2) the multiple different preparation hybridoma of the present invention and the approach of antibody are provided.
For the present invention, L/1C2 antibody only is illustrative, all can with the antigen reactive antibody of L/1C2, no matter its source species or immunoglobulin class or hypotype classification (comprising IgG, IgA, IgM, IgE and IgD) how, all comprise within the scope of the invention.The present invention also comprises the L/1C2 antigen-binding fragments of antibodies.With L/1C2 antigen bonded ability be a general characteristic of antibody of the present invention.
As discussed above, antibody of the present invention can make up and separate with the following step, i.e. immunity, preparation hybridoma, then identify can to tumor cell line reaction and its healthy tissues distribution antibody similar to L/1C2 antibody.But the present invention also provides a kind of method of identifying antibody of the present invention, and this method need not to measure the reactive behavior of antibody and massive tumor and healthy tissues.The L/1C2 antibody that antibody of the present invention can utilize L/1C2 clone to produce is identified by immunoprecipitation and competitive binding experiment.
As described in example 2, obtain similar mobility spectrum when carrying out immunoprecipitation with the L/1C2 monoclonal antibody, can utilize these collection of illustrative plates to identify antigen.Can utilize following method to confirm qualification result: from cell extract, to remove this antigen with excessive a kind of antibody, and observe another antibody and whether can not from handled extracting solution, go out antigen by immunoprecipitation.In addition, under antibody and same epitope or closely-related epitope bonded situation, each antibody will be competed each other with L/1C2 is antigenic and combine.Competitive binding experiment is described in example 4.
With proteolytic ferment such as papoid and pepsin antibody preparations, produce some antibody fragments, comprise the Fab and the F(ab ' that have kept antigen-binding activity)
2Fragment.Therefore, can utilize these enzymes to handle antibody of the present invention, thereby produce L/1C2 binding fragment of the present invention.The preparation of antigen-binding fragments of antibodies of the present invention and their description of uses in treatment are in example 17.The L/1C2 antigen-binding fragments of antibodies is specially adapted to treatment plan of the present invention.
This area professional will know that the antigen binding domain of antibody of the present invention and antibody fragment are key features of the present invention.L/1C2 hybridoma of the present invention is the preferred source of the DNA of a kind of like this antigen binding domain of code book invention.Can this DNA be linked to each other with any required amino-acid residue DNA in proper order of coding by recombinant DNA technology, thereby generate new " hybridization " or " chimeric " DNA sequence, this DNA sequence a kind of hybridization of coding or chimeric protein.Can obtain chimeric antibody of the present invention by this way, in these antibody, final a part of antibody sources is in species, and another part derives from another species.But the present invention also comprises the chimeric molecule of any L/1C2 of containing antigen binding domain.
Comprise the antibody of the present invention of L/1C2 antibody, thereby can be used for whether having cancer to exist in the immunoassay diagnosis people tissue sample.Can utilize L/1C2 antibody of the present invention, whether have cancer to exist in the biopsy samples of evaluate patient and the postmortem sample.Can express the antigenic cancer of L/1C2 to identify with some detection moiety mark antibody of the present invention, these groups comprise fluorescent mark, enzyme labelling and radio-labeling.Being used for detection moiety of the present invention comprises as fluorescently-labeled fluorescein, as the peroxidase of enzyme labelling with as radiolabeled iodine-125.
Other can be used in the compound that fluorescent mark of the present invention includes, but is not limited to rhodamine, phycoerythrin and some other emitting fluorescence energy.Other can be used in enzyme labelling of the present invention and include, but is not limited to glucose oxidase and alkaline phosphatase.Other can be used in radio-labeling of the present invention and include, but is not limited to iodine-131 and indium-111.This area professional should know clearly that above-mentioned these marks only are that explanation can be used for not isolabeling of the present invention.Can with the antigen reactive antibody of L/1C2 can also be by combining and derivatize (example 7) with vitamin H, promptly can be used for behind adding and fluorescent mark, enzyme labelling or radio-labeling bonded avidin compounds in the application of panimmunity chemistry and immunohistology, above-mentioned antibiont protein compound can be detected owing to combine marker.Method with antibody test of the present invention and diagnosis cancer is illustrated in example 6.
The characteristic of L/1C2 antibody comprises the general characteristic of antibody of the present invention, can combine with L/1C2 antigen.By hybridoma cell line L/1C2(ATCC HB9682) special characteristics of the L/1C2 reactive antibody that produces comprises following physical property: such as example 8 mensuration, be mouse IgG3 homotype; Produce (SDS-PAGE that sees Fig. 1 analyzes) as the secreted unique immunoglobulin (Ig) of L/1C2 clone (being ATCCB9682); Solubleness is 10mg/ml in PBS.
The practicality of L/1C2 antibody in treatment adds their confirmation with the cellular toxicity drug conjugates of this antibody, and further confirms, even the antibody of unmodified also has external activity.The ability of L/1C2 antibody inhibiting tumor cell in-vitro growth is summarized in table 3.The external test method of growth of tumour cell is specified in example 9.
Table 3
The tumor cell extracorporeal growth restraining effect
1
Concentration C PM+/-S.E. % changes P
L/1C2
100μg/ml 57，144 +/- 7，242 -92 .001
10 424，406 +/- 98，658 -41 .05
1 595，578 +/- 109，209 -19 ns
0 736，567 +/- 19，026
L4/KS
100μg/ml 698，823 +/- 81，625 -8 ns
10 656，286 +/- 43，969 -13 ns
1 709，928 +/- 12，1271 -6 ns
0 758，271 +/- 18，931
Bleomycin sulfate
10μg/ml 89，906 +/- 13，685 -86 .01
0 648，327 +/- 109，017
1What provide is
3The H-leucine mixes the count per minute (CPM) in the T222 squamous cell cancer target cell.A large amount of sample requirements in this mensuration use a more than tissue culture ware.Therefore, every kind of reagent is all made comparisons with the one group of negative control aperture of himself and is calculated the inhibition percentage.The P value utilizes Student t check to determine.In this mensuration, introduce bleomycin and make positive control.
Table 3 has provided the comparison of the ability of L/1C2 antibody and L4/KS antibody inhibiting tumor cell in-vitro growth.Specifically, table 3 has illustrated that unmodified L/1C2 antibody that concentration is low to moderate 10 μ g/ml significantly suppresses the ability of L/1C2 antigen positive tumor cell line T222 growth.With KS1/4 antigen (Varki et al., Cancer Res., 44: 681,1984) bonded L4/KS antibody (Starling et al., J.Cell Biochem., Supplement 11B: 192,1987) also be expressed in the T222 tumour cell and fasten.Opposite with L/1C2 antibody, L4/KS antibody does not suppress the effect of T222 cell growth.
New ability for the L/1C2 antibody inhibiting tumor cell of understanding unmodified is grown experimentizes to determine complement combination and antigen internalization role.Utilize serum to carry out example 9 described mensuration, this serum heats 30 minutes down to cause the heat inactivation of complement activity in 56 ℃.This result of experiment shows that the combination of complement is not the reason that causes the viewed cytotoxicity level of unmodified antibody.These data are that with the consistent part of table 3 data L4/KS is a kind of IgG2a phenogen, and this is a kind of known antibody subtype that plays an important role in the complement combination.
For estimating L/1C2 antibody and antigen positive tumor cell line bonded result, example 10 and 11 described assay methods have been set up.What example 10 was set forth is, utilize fluorescein-labeled can with the antigen reactive antibody of L/1C2, estimate L/1C2 antibody in the kinetics that combines the back internalization with the L/1C2 antigen of cell surface.Fig. 3 has provided the result who utilizes this method to detect the antibody internalization.It should be noted that among Fig. 3 that in the time of 0 minute (figure A), the peculiar bright ring sample of film topography shape fluorescence clearly.On the contrary, after 105 minutes (figure B), encircle the sample fluorescent weakening, and the cell periphery there is tangible bright fluorescence beam in insulation under 37 ℃.At last, (figure C) no longer include bright ring sample fluorescence, but fluorescence become obviously in the cell in the time of 135 minutes.Cell inner dyeing shows that the internalization of L/1C2 antibody is and L/1C2 antigen bonded result.
The result of example 11 described mensuration shows,
125The L/1C2 antibody of I mark is by internalization.Confirm that with radiolabeled antibody this derives from the internalization data of example 10 fluorescence techniques, to get rid of cell surface bonded L/1C2 antibody by the crosslinked any effect that in inducing the internalization process, may play of second kind of reagent of glimmering actinic (anti-mouse immuning ball protein).Table 4 has provided the result of this mensuration.The data of table 4 confirm that clearly L/1C2 is combining the back internalization with L/1C2 antigen.
Table 4
With
125I-L/1C2 antibody is estimated the internalization of L/1C2 antibody
0℃ 37℃
Mean value+/-standard error mean value+/-standard error
125I-L/1C2 56，770+/-3，069 56，746+/-1，734
Total cpm
1
Can not be dissociated 11,398+/-859 5,514+/-64
Counting
2
1 125Total cpm of I-L/1C2 measures for the gross activity that links with cell, and it should both comprise cell surface
125I-L/1C2 also comprises internalization
125I-L/1C2.
2Can not dissociated counting be internalization
125The a kind of of I-L/1C2 measures, and be this
125I-L/1C2 can destroy the antigen/antibody combination owing to internalization is difficult for being dissociated by low pH glycine buffer and sort buffer liquid is known.
The cytostatic mechanism of L/1C2 antibody is still indeterminate, but according to estimating that L/1C2 antigen may be a kind of growth factor receptors.Someone has reported the relevant internalization that obtains with anti-epidermal growth factor (EGF) receptor antibody and the class likelihood data (Mosui of cell growth inhibition, et al., Cancer Res.45:5592,1986), but the possibility of L/1C2 antibody and EGF receptors bind has been got rid of in the analysis that CBA and clone distribute.
This area professional will recognize that antibody is combining the meaning that internalization takes place in the back with tumor-cell antigen.The validity of immune conjugate (antibody-cytotoxic drug) depends primarily on the ability that it hits tumour cell, next depends on that it provides the cytotoxic agents of toxic forms or discharges cytotoxic agents at tumor locus, thereby makes the ability of cytotoxic agents killing tumor cell.Antibody and internalization after tumour cell combines have caused in the cell of cytotoxic agents to discharge.Therefore release will be a preferred cytotoxic agents release system in this cell of cytotoxic agents.
Now successfully many different cytotoxic drugs have been connected on the antibody.Someone has summarized method (Blair et al., J.Immunol.Mothods 59: 129,1983 that connect cytotoxic drug and antibody; Ghase et al., Methods in Enzymology, 93: 280,1983).The preferred monoclonal antibody of the present invention-cytotoxic drug binding substances (immune conjugate) comprises the immune conjugate that those are produced by the hydrazine derivative reaction of oxidized form L/1C2 antibody and vinca alkaloids.The preparation method of these potent antitumor immune binding substancess presses disclosed European patent bulletin 247,792 on December 2nd, 1987 substantially, and its disclosure is classified this paper reference as.European patent bulletin 247,792 discloses the construction process of the immune conjugate that contains L/1C2 antibody.Example 13 of the present invention discloses the method that the vincaleucoblastine hydrazides is connected with L/1C2 antibody.Can announce 247 with reference to European patent; 792 preparations, combining method, and going through of being done of immune conjugate preparation with regard to the vincaleucoblastine intermediate, but this patent not have to discuss the L/1C2-4-deacetylation-vincaleucoblastine-3-carboxyl hydrazides that is used as preferred therapeutic scheme of the present invention herein.
In the mouse heteroplastic transplantation model described in the example 12, with L/1C2-4-deacetylation vincaleucoblastine-3-carboxyl hydrazides (L/1C2-DAVLBHYD) or free 4-deacetylation vincaleucoblastine-3-carboxyl hydrazides (DAVLBHYD) or as the PBS intravenous administration of thinner in nude mice.With L/1C2-DAVLBHYD, DAVLBHYD or 11st, 15,17,22, the 25 and 29 day processing mouse of PBS after the tumour heeling-in, obtain data shown in Figure 4.
The data acknowledgement of Fig. 4 L/1C2 antibody strengthen the ability of DAVLBHYD result of treatment.Use the L/1C2-DAVLBHYD binding substances in the experiment, obtained the data of Fig. 4 by this experiment.After measured, the average knot binding ratio of L/1C2-DAVLB immune conjugate is 5.7: 1(DAVLBHYD: L/1C2).The dosage of the following stated refers to the DAVLBHYD content of single therapy.When using DAVLB with the form of L/1C2-DAVLBHYD immune conjugate, 1.0mg/kg and the dosage of 0.5mg/kgDAVLBHYD (figure A) just causes the degeneration of tumour, and the free DAVLBHYD(figure C of same dosage) when treating, only block growth of tumor.Obviously as seen, when vincaleucoblastine content was 1.0mg/kg, the L/1C2-DAVLBHYD immune conjugate can be eradicated the T222 cancer in the last figure of Fig. 4.Comparison shows that of relative effectivenes, in whole experiment, under the concentration of 1.0mg/kg and 0.5mg/kg, L/1C2-DAVLBHYD is better than free DAVLBHYD.The data that provide of figure B show, if DAVLBHYD with a kind of not with T222 cell bonded antibodies, it is invalid then retardance T222 cell to be grown.Can with the antigen reactive immune conjugate of L/1C2, as L/1C2-DAVLBHYD, than the free drug of Isodose level or can not more can suppress the T222 tumor cell line effectively with the antigen reactive immune conjugate of L/1C2.Above-mentioned data declaration the therapeutic action of L/1C2 antigen-binding immune conjugate.
Further research has been established L/1C2 antibody and be can be used as and a kind ofly discharge the carrier that other born of the same parents are poisoned compound to L/1C2 antigen positive tumour cell.Some immune conjugates have been made up, wherein have methotrexate and L/1C2 antibody coupling, coupling method has plenty of the hydrazides of being discussed in the example 13 and connects key, also has plenty of and utilizes some linking group to form a hydrolyzable bridged bond between L/1C2 antibody and methotrexate.The methotrexate method that is methotrexate-γ-hydrazides of deriving is discussed in the A of example 14 part.Substantially the method by example 13 makes methotrexate-γ-hydrazides and oxidized form L/lC2 antibodies.With the 4-deacetylation vincaleucoblastine-3-carboxyl hydrazides of methotrexate-γ-hydrazides replacement example 13, make L/1C2-methotrexate immune conjugate can be used for some treatment and use.Estimate the vivo antitumor activity of these immune conjugates with the bare mouse different species transplantation model, details is seen example 12.In this experiment, after the tumour heeling-in the 3rd, 6 and 9 day is with L/1C2 methotrexate-γ-hydrazides, methotrexate or diluent treatment mouse.Listed data are represented to suppress per-cent with respect to viewed growing tumors to only with the diluent treatment animal time in the following table 5.
Table 5
L/1C2-methotrexate-γ-hydrazides and trip
External activity from methotrexate compares
Dosage tumor suppression per-cent in methotrexate content
L/1C2-methotrexate-γ-hydrazides 6mg/kg 97
3mg/kg 99
1.5mg/kg 94
Free ammonia methopterin-A 6mg/kg 32
3mg/kg -4
1.5mg/kg 23
The presentation of results of table 5 when methotrexate is hit tumor locus as L/1C2 antigen reactivity immune conjugate, it is renderd a service greatly increases.280 and the 370nm place do the dual wavelength ultra-violet analysis, the binding ratio that records this immune conjugate is every mole of L/1C2 antibody 5.1 moles of ammonia methopterin-As.
Further description of test L/1C2 antibody and its Fab discharge the purposes of cytotoxic agents, these cytotoxic agents are by hydrolyzable linking group and L/1C2 or its F(ab ')
2The fragment covalency links to each other.Example 14 to 17 discloses the preparation and the evaluation method of these immune conjugates.
Data acknowledgement listed above L/1C2 antibody or its Fab with various interconnection technique with have therepic use after cytotoxic drug is connected.This area the professional will recognize that, combining with L/1C2 antigen is a general characteristic of antibody of the present invention; Different with the antibody of ATCC B9682 generation is that these antibody of the present invention can also play and make cytotoxic agents hit the effect of L/1C2 antigen positive tumour.Should consider some variation of the affinity of L/1C2 antigen and antibodies of the present invention, in general, the antibody affinity is high more, and antibody just can make cytotoxic agents hit in-vivo tumour more effectively.Provide the immune conjugate that contains L/1C2 antigen reactivity antibody, L/1C2 antibody, vinca alkaloids derivative and methotrexate, just in order to help understanding to Antybody therapy purposes of the present invention.The application of antibody of the present invention in treatment never only limits to those and is used for illustrating cytotoxic agents of the present invention.Other can be used for the cytotoxic agents that the present invention treats application and comprise, for example, adriamycin, ricin, ricin A chain, dipteral insect toxin, Pseudomonas exotoxin, lampwick alcohol (Scirpenol), diacetoxy lampwick alcohol, α amanitin, ametycin, abrin, gel element (gelonin), pokeweed antiviral protein, 5 FU 5 fluorouracil etc.Other many methods of attachment, as United States Patent (USP) 4,671,958 and EPO bulletin 243,929 disclosed methods, also can be used to make cytotoxic agents to link to each other with antibody of the present invention, these methods are classified this paper reference as.
Use L/1C2 antigen-binding monoclonal antibody of the present invention and carry out the in-vivo tumour imaging also within the scope of the invention.The strong ⒌ Suan Chu river which rises in the northeastern part of Anhui Province ㈨ dysentery ㈩ spinulose tree fern ⑴ Zhen ⒉ ⒁ ㈩ bery used as a personal name in ancient times ㈩ umbrella ⒐ ㈩ frame river which rises in the northeastern part of Anhui Province ⒘ of the towering marquis huge legendary turtle of same phoenix ⑶ Α that the coordination that can be used to radio-labeled antibody of the present invention have following element lives the ㈩ beer
Radio isotope, for example above-mentioned isotropic substance that is used for tumor imaging when linking to each other with L/1C2 antigen-binding antibody of the present invention, can be used in the treatment application.Therefore, by the application of L/1C2 antigen-binding antibody-therapeutical agent that the radio isotope immune conjugate is formed also within the scope of the invention.
Example
For ease of understanding the present invention, provide following limiting examples.
Example 1
The structure of the reactive hybridoma cell line of L/1C2
A. immunity
The MONOCLONAL ANTIBODIES SPECIFIC FOR method is introduced (Nature 256: 495,1975) by Kohler and Milstein, and this method now has been the mature technology in this area.One piece of newer summary (Gaflre and Milstein is arranged, Methods in Enzymology, 73:3-46,1981, Langone and Van Vunakis, ed., Academic Press, New York) some that summarized original method improved, and describes required instrument and reagent in detail, has been described in detail the step of preparation monoclonal antibody.What immunogenic selection only limited to clone, tumor sample or they contains the antigenic any composition of L/1C2.
With the clone USCLS-1(Fernstein of tissue culture method breeding from squamous cell cancer, et al., Cancer Research 46:2970-2977,1986), and as the antigenic source of L/1C2.USCLS-1 clone only is illustrative, and the antigenic clone of any expression L/1C2 (seeing Table 1) can adopt.Get the young BALB/C mice of having grown up, intraperitoneal injection 5 * 10 weekly
6Individual USCLS-1 cell injected for 4 weeks altogether.The 4th injection is after 21 days, with 5 * 10
6Individual USCLS-1 cell carries out immunity again to mouse.Concerning this method, mouse only is illustrative, and the professional knows what restriction the species as the immunocyte source are not had.Known other species comprise that people, rat and hamster are comparatively suitable, but any in theory species can use, and comprise rabbit, goat, sheep, pig, chicken and monkey.Utilize trypsinase EDTA(Gibco, 3175 Staley Road, Grand Island, New York 14072) facture, reclaim the USCLS-1 cell as L/1C2 immunogenicity of antigens source from tissue culture.
B. the preparation of splenocyte
Last immunity is after 5 days, with the BALB/C mice broken end of immunity and use alcohol flushing.Aseptic collection spleen also places an aseptic little culture dish, for the usefulness of splenocyte preparation.Choose out spleen, splenocyte is discharged in the Eagle substratum (DME-Gibco) of ice-cold DulbeccoShi modification.By suction cell mass is disperseed, and cell is moved into a centrifuge tube, will place 5 minutes among this Guan Zaibing.Discard sediment, cell suspending liquid is moved in second centrifuge tube.With centrifugal 5 to 7 minutes sedimentation splenocytes of 200g.The cell precipitation that obtains is suspended in the ice-cold 0.17M NH of 5ml
4Among the Cl, placed 10 minutes, to promote the fragmentation of cell on ice.Add the ice-cold DME of 5ml, then with this mixture centrifugal 5 to 7 minutes with 200g.After the sucking-off supernatant liquor, will remove erythrocytic splenocyte precipitation resuspending and in 10ml DME, wash.
C. myeloma cell's preparation
Select HL-1
TMFriendly myelomatosis-653 cell (Ventrex Laboratories, P.O.Box 9701, Portland, Maine 04013) be used for merging with the reactive splenocyte prepared product of USCLS-1.These cells derive from Ventrex, and preserve by supplier's specification sheets.Merging a few days ago, the myeloma cell is moved into 75cm
2In the tissue culture flasks, make it reach the logarithmic phase growth, this is very important for successfully generating hybridoma.
Though it is HL-1 that pair cell merges preferred myelomatosis
TMFriendly653 cell, but some conventional myeloma cell lines from mouse of having set up also can be used for purpose of the present invention, these cells comprise,
P3-X63-Ag8-U1（P3U1），SP2/O-Ag14（SP-2），
P3-X63-Ag8-6.5.3（X63.6.5.3），p3-X63-Ag8（X63），
P3-NS-1-Ag4（NS-1），MPC11-45.6 TGI.7（MPC-11），and
S194/5XXO.BU1(S194) (see Gaflre and Milstein, 1981, in
“Methods in Enzymology”Vol.73B，Langone and Van Vunakis，ed.）。
D. cytogamy
With following method washing splenocyte and myeloma cell: under 4 ℃ centrifugal 5 minutes with 200g, sucking-off supernatant liquor then, with the cell resuspending in DME.After DME washing 3 times, pair cell is counted, and is estimated as motility rate with trypanblue exclusion method simultaneously.Here used cell number one speech only refers to viable cell (not by the painted cell of trypan blue).In the 50ml centrifuge tube 3.0 * 10
7Add 1.5 * 10 in the individual splenocyte
7Individual myeloma cell.With cell under 4 ℃ with 200g centrifugal 5 minutes, as far as possible fully the sucking-off supernatant liquor did not lose the cell in the precipitation again.Patting centrifuge tube then gently makes cell precipitation loosening.Add 1.5ml down in 37 ℃ and merge medium (compound method as follows), stir content gently, centrifuge tube was left standstill 30 seconds.Drip then to be preheated to 37 ℃ DME and to accompany by gently and stir, make the volume of content slowly reach 20ml.The DME that adds 37 ℃ of 30ml again.Must be noted that during the diluted mixture thing and avoid smashing cell mass.With fusions with 200g centrifugal 5 minutes, resuspending contained the HL-1 of 20% foetal calf serum (Gibco) and 1 * HAT in 60ml carefully then
TMIn the substratum (Ventrex).
Select splenocyte/myelomatosis hybrid cell with HAT.HAT is the mixture of xanthoglobulin, aminopterin and thymidine.Below provided HAT and PEG4000(and merged medium) compound method of prepared product.
HAT(100X) amount/25ml
Xanthoglobulin (1000 μ M) 34mg
Aminopterin (100 μ M) 11mg
Thymidine (300 μ M) 18.25mg
During preparation, 3 kinds of materials are dissolved in respectively among 1 to 5 1N NaOH, they are mixed, washing test tube and add to volume with DME is 25ml.Use substratum with 1: 100 dilution HAT during use.HAT also can buy from Gibco.
Merging medium can prepare with the following method.With 20g PEG 4000(J.T.Baker Chemical Co., 222 Red School Lane, Phillipsburg, New Jersey 08865) be contained in and carry out autoclaving in the 100ml bottle.Adding 28ml contains the aseptic DulbeccoShi PBS(Gibco of 15%DMSO), mix the back and store down in 4 ℃.
E. the preparation of feeder cell
Be expelled to the intraperitoneal of BALB/C mice with No. 18 syringe needle DME that 10ml is ice-cold.Stir the abdominal cavity, take out the abdominal cavity washings.Abdominal cavity cell is placed ice-cold centrifuge tube, centrifugal 5 minutes with 250g.After the sucking-off supernatant liquor, with the cell resuspending in the ice-cold HL-1 substratum (Ventrex) of 2ml and be placed on ice.
F. the tissue culture of hybridoma
Feeder cell (abdominal cavity cell that washes) is merged and mixes with the cytogamy mixture.Every hole adds 2 cell mixtures in 96 hole tissue culture plate.Utilize 37 ℃ of moist incubators to add and contain 5%CO
2Air preserve the cytogamy product.Add the HL-1(Ventrex that contains 20% foetal calf serum (Gibco) and 1 * HAT) make the volume of all apertures all increase to 200 μ l.
G. the selection of antigen-specific hybridoma
When in 96 hole flat boards, obviously macroscopic clone occurring, get duplicate samples such as 50 μ l and estimate its antigenic response activity.Utilize radioimmunoassay (RIA) to measure supernatant liquor and USCLS-1(L/1C2 positive target cell system) and M21(L/1C2 negative cells that derives from melanoma be) combine (Morton et al., Surgery 64(1): 233-240,1968), details are as follows for this method.Also can adopt other L/1C2 positive or negative target cell system, comprise the clone in the table 1.
By using CPEG(136mM NaCl, 2.7mM KCl, 8mM Na
2HPO
4, 1.5mM KH
2PO
4, 0.5mM EDTA, 5.6mM glucose, pH7.3) handled 15 minutes down, and use PBS(Gibco in 37 ℃) washing, collection USCLS-1 and M21 clone from tissue culture medium (TCM).With 1.5 * 10
5Individual cell is placed in the 96 hole elastic plates, and these flat boards are in advance with the following method with poly-L-Methionin bag quilt.In each aperture of flat board, add the solution of the poly-L-Methionin hydrobromate of 50 μ l 1mg/ml (St.Louis, Missouri 63178 for Sigma Chemical Company, P.O.Box14508) in PBS.Should under room temperature, be incubated at least 45 minutes by flat board then, but also can store down at 4 ℃.Before adding cell, flat board is washed twice with PBS.With the still unconjugated cell of PBS flush away.The cell that stays is fixed with the PBS solution of 0.1% glutaraldehyde.Seal unreacted glutaraldehyde with 0.1% glycine, with flat board with containing 10% foetal calf serum and 0.05% NaN
3Phosphate buffered saline(PBS) washing and be stored in this solution.Phosphate buffered saline(PBS) (PBS) is for containing the 0.01M sodium phosphate of 0.15M NaCl.
Carrying out RIA with following method measures: at room temperature (about 22 ℃) are incubated 1 hour with the hybridoma supernatant liquor with test clone.Thorough washing is removed unconjugated antibody, add with Chloramine T method (Herzenberg and Herzenberg, Handbook of Experimental Immunology, 1978, D.M.Weir, ed.Blackwell Scientific Publications, Oxford) 100 of mark, 000cpm
125The anti-mouse IgG of I-rabbit is incubated 1 hour again under room temperature.Wash each aperture with PBS, utilize radioautograph and gamma counter to measure the amount of residual activity in every aperture.The radioautographic analysis process comprises, 96 hole flat boards are placed on the X-ray film, observes the little hole count that contained radioactivity is enough to make exposure.The autoradiographic time shutter is not waited from 6 hours to spending the night.Generation can be with USCLS-1 reaction but can not be considered to desired clone with the hybridoma clone of the antibody of M21 reaction, and it is further analyzed.L/1C2 hybridoma cell line ATCC #B9862 can react with USCLS-1, can not react with M21.Use Coulter
(FL) the subclone instrument is pressed the manufacturer's recommended method with L/1C2 subclone twice, to guarantee the monoclonicity of L/1C2 hybridoma cell line for Coulter Electronics, Hialeh.
H. the mensuration of being discerned by the L/1C2 reactive antibody from the clone of tumour
L/1C2 is the illustrative hybridoma of clone of the present invention.Estimate the reactive behavior of the L/1C2 antibody of hybridoma generation thus to one group of tumor cell line.Screen with the indirect immunofluorescence assay method.The mensuration mixture comprises: a kind of target cell system that needs mensuration whether to have new L/1C2 tumor associated antigen; Hybridoma supernatant liquor (source of L/1C2 reactive antibody); A kind of usefulness is fluorescein-labeled and be specific to the second antibody reagent of mouse immuning ball protein.The second antibody reagent that is suitable for can have been bought from many places, as Jackson ImmunoResearch Laboratories, Inc., P.O.Box 683, Avondale, Pennsylvania 19311, and Miles Research Products, Miles Laboratories, Inc., 1121 Myrtle, Box2000, Elkhart, Indiana 46515.
Utilize condition well known in the art to cultivate target cell system with tissue culture method.Before the mensuration, from the substratum that is added with trypsinase/EDTA(Gibco), take out cell.In DME, wash 5 * 10 by centrifugal and resuspending
6Individual cell, and cell and hybridoma supernatant liquor were reacted 1 hour in 4 ℃ in 12 * 75mm test tube.Undertaken centrifugal and unreacted antibody in the flush away cell by lower berth one deck foetal calf serum.With the target cell resuspending in the 2nd reagent sheep F(ab ')
2Anti-mouse (IgG+IgM) FITC(fluorescein isothiocyanate) in the binding substances (derive from Tago Laboratories, Burlingame, CA 94010), and finally uses with dilution in 1: 50.Test materials was kept under 4 ℃ 1 hour.Undertaken centrifugal and target cell and unconjugated second reagent are separated by lower berth one deck foetal calf serum.Sucking-off serum is with Hanks balanced salt solution (HBSS, Gibco) washed cell.With cell again with the HBSS washing once, resuspending remains on 4 ℃ down so that observe with ultraviolet fluorescence microscopy in tissue culture medium (TCM).Get the equal portions cell sample with propidium iodide(10 μ g/mlHBSS solution) to redye, the cell that further confirms antibody staining is viable cell (propidium iodide optionally dye dead cell).The viable cell that demonstrates classical surface ring sample fluorescence is considered to the L/1C2 antigen positive.
In addition, also available Epics-Coulter
Mark IV
Cytoanalyze (Coulter Electronics) utilizes the fluorescence of method assess sample well known in the art.The method of other screening tumor response sex clones is specified in (Galfre and Milstein, Methods in Enzymology, 73:3-46,1981, Langone and Van Vunakis, ed., Academic Press, New York) in the document.
I. utilize antibody evaluation human pathology sample of the present invention
Utilize avidin-biotin technology of revising, distribute with immunoperoxidase procedure evaluation of tissue and tumour antigen.Freezing tissue piece is cut into 4-6 μ m size, is placed on the slide glass with gelatin bag quilt, air-dry, in acetone, fix 5 minutes.After adding 5% horse serum pre-incubation, with tissue block successively with L/1C2 antibody, the anti-mouse Ig(Vectorlabs of biotinylated horse, Burlingame, CA), the mixture of avidin and horseradish peroxidase is incubated, between these incubation step with the phosphate buffered saline(PBS) pH7.2-7.4 washing that contains 1% bovine serum albumin(BSA).The optimum dilution degree of all the components all passes through " chessboard " volumetry commonly used in this area and is determined by experience.With 3,3 '-the diaminobenzidine colour developing, with the tissue block haematoxylin redyeing.The result is summarized in table 2.
The J.L/1C2 purifying antibody
Behind 5 microns membrane filtrations (Millipore), ascites fluid is added to A Protein S epharose post (Pharmacia Fine Chemicals, Division of Pharmacia Inc., 800 Centennial Avenue, Piscataway, New Jersey 08854) on, antagonist carries out affinity purification.Lavation buffer solution is 0.01M sodium phosphate (pH8.0), and (pH3.5) carries out gradient elution with the 0.1M sodium phosphate buffer.Each component that wash-out goes out (pH7.4) neutralizes rapidly with 1M Trizma damping fluid (Sigma), and adds 0.15M NaCl(PBS with 0.01M sodium phosphate (pH7.4)) dialyse.Antibody preparations is filtered by 0.22 micron filter membrane (Bedford, Massachuetts 01730 for Millipore Corporation, Ashby Road) and sterilize, store standby down in 4 ℃.
The preparation of K.L/1C2 antibody
The L/1C2 hybridoma produces 5-6mg/ml antibody as ascites well-grown in feeding the BALB/C mice body of pristane.Utilize the A protein chromatographic and the L/1C2 productive rate of purifying in the 70-90% scope, through SDS-PAGE(Laemmli, Nature(London) 227: 680,1970) and Coomassie blue stain (Figure 1A) identify that gained purity is greater than 90%.The L/1C2 antibody of purifying has fabulous solvability in PBS, it is above and can be owing to precipitation is lost that concentration therein can reach 10mg/ml.
Example 2
L/1C2 is antigenic qualitative
Utilize radiolabeled 5637 cells (ATCC #HTB9) to measure by the sedimentary antigenic character of L/1C2 antibody mediated immunity.5637 clones are a kind of illustrative clone of L/1C2 antigen positive cancer, and any L/1C2 positive cell line in the table 1 all is suitable for.With tissue culture method cell cultures is converged to 50-75%, the cell that will adhere to is with aseptic phosphate buffered saline(PBS) (PBS) pH7.4(Gibco) wash twice.With 1mCi
3The H-glycosamine (New England Nuclear, 549 Albany Street, Boston, Massachusetts 02118,617-482-9595) dilutes in substratum, is added in the culturing bottle.After 24 hours, the cell of metabolic marker is washed twice with cold PBS, then with containing 0.02% sodium azide, 2mM phenylmethylsulfonyl fluoride, 1%Nonidet
P-40(Sigma) and the PBS of 0.1% dodecyl sulphate extracted on ice 20 minutes.With 15, centrifugal 15 minutes of 000g, with 100, centrifugal 1 hour of 000g (centrifuging temperature all is 4 ℃), then that the freezing storage of radiolabeled supernatant extracting solution is standby.Before the use, with extracting solution and A Protein S epharose
(Sigma) be incubated together to remove non-specific junction mixture.Followingly carry out indirect immunoprecipitation: 1 * 10
7Add 50 μ g L/1C2 antibody in the cpm labeled cell extracting solution, this mixture is incubated 4 hours down in 4 ℃.In pipe, add IP damping fluid (PBS, 1% BSA, 0.1% Nonidet P-40 then
) in the A Protein S epharose that washed of 100 μ g20%
(Sigma), and then gently vibrated 4 hours.It is centrifugal twice to add the IP damping fluid, and it is centrifugal twice only to add PBS again, washs antigen, antibody and A Protein S epharose with this
Mixture.After the last washing, analyze throw out with SDS-PAGE.
The antigen that immunoprecipitation is gone out carries out the used standard protein of molecular weight determination and comprises: N,O-Diacetylmuramidase (14,400), the soybean pancreatin inhibitor (21,500), carbonic anhydrase (31,000), ovalbumin (45,000), bovine serum albumin(BSA) (66,200), phosphorylase B (92,500), beta-galactosidase enzymes (116,250), myosin (200,000).Used molecular weight marker obtains (Richmond, California 94804 for Bio-Rad Laboratories, 2200 Wright Avenue) with kit form.
Contain slab gel through isolating sample with Coomassie blue stain, show molecular weight marker, then gel is carried out radioautograph, show mark antigen (Fig. 1, B).
Example 3
The preparation of L/1C2 antibody
Can be from ATCC(Rockville, MD) obtain the freeze-drying bottle of L/1C2 hybridoma, registration number is ATCC #B9682.In 37 ℃ of water-baths, melt content in the bottle, rotate this bottle simultaneously promoting fast and evenly thawing, thereby be recovered to viable cell.Cell suspending liquid is diluted with 1: 2 with balanced salt solution (BSS-Gibco), and lower berth serum layer is centrifugal with 200g, thereby isolates cell from refrigerant.Behind the material above the sucking-off cell precipitation, collecting cell, with this area commonly used be added with serum and the dilution of antibiotic tissue culture medium (TCM), and under standard conditions (37 ℃, 5%CO
2) carry out cell cultures and make its growth and breeding.In cell cultivation process, preferably keeping cell concn is 1 * 10
5-7 * 10
5Individual cell/ml, but the variation of some appropriateness also is complete acceptable.
Can from tissue culture, the amount with μ g/ml reclaim L/1C2 antibody.Also can make L/1C2 in the rodent body, be established as ascites tumour and produce antibody with higher productive rate.The preparation of antibody and collection method are specified in (Gaflre and Milstein in one piece of fabulous summary, Methods in Enzymology, 73B:43-45,1981, Langone and Van Vunakis, ed., Academic Press, New York).
Example 4
Measure and antigen bonded antibody of the present invention
When measuring the antibody that reacts with L/1C2 antigen positive target cell system and whether combining, competitive binding experiment can be utilized, also immunoprecipitation analysis can be used with L/1C2 antigen.Competitive binding experiment is known to the professional.In this class experiment, in the mensuration of certification mark form L/1C2 antibody, introduce the specific antibody of a kind of unknown antigen.The inhibition of L/1C2 antibodies just shows that unknown antibody combines with L/1C2 antigen.
A. competitive assay
In competitive radioimmunoassay (RIA), as described in example 1, prepare and separation antibody with A protein chromatographic method, and press Tsu and described (the Selected Methods in Cellular Immunology of Herzenberg, P.373-380, ed.Mishell and Shiigi, W.H.Freeman and Company, San Francisco) use
125I carries out radio-labeled.Any all is suitable target cell concerning competitive RIA as the listed antigenic clone of expression L/1C2 of table 1.With the antigenic clone of tissue culture method culture expression L/1C2, up to reaching desired cell number.Handle by CPEG and from cell matrix, to discharge cell and wash with PBS.With 1.5 * 10
5Individual cell is placed in the 96 hole elastic plates, and elastic plate is in advance with poly-L-Methionin (Sigma) bag quilt.With the cell that the PBS flush away does not adhere to as yet, the cell that stays is fixed 5 minutes with 0.1% glutaraldehyde (2/hole) under room temperature.Seal unreacted glutaraldehyde with 0.1% glycine, with containing 10% foetal calf serum and 0.05%NaN
2PBS washing elastic plate and elastic plate is kept in this solution.
The preparation method of target cell flat board sees document (Starling et al., Cancer Research 46(1): 367-374,1986; Starling et al., Journal of Supramolecular Structure 11(4): 563-577,1979), these documents are classified this paper reference as.Determine by experience
125The optimum dilution degree of I-L/1C2 antibody, after the adding hybridoma supernatant liquor, this mensuration system just can be used as contention system.
The another kind of utilization
125I-L/1C2 and the method for measuring of being at war with property of hybridoma sample see document (Tsu and Herzenberg, Selected Methods in Cellular Immunology, ed., Mishell and Shiigi, p 390-394, W.H.Freeman and Company, San Francisco).
B. the immunoprecipitation analysis of antigen-specific
Also can utilize the immunoprecipitation of radiolabeled cellular component determine a kind of antibody whether with L/1C2 antigen-reactive of the present invention.Carry out qualitative with example 2 described methods to antibody of the present invention institute bonded antigen.Compare carrying out the resulting mobility spectrum of immunoprecipitation, whether have antigen identity disclosing two kinds of different antibody with L/1C2 antibody or another kind of antibody.The research of competitive binding experiment and immunoprecipitation is combined, provide another kind of definite antibody whether with the antigen reactive means of the present invention.
Example 5
With the antigen reactive polyclone of L/1C2
The preparation of antibody
At soluble antigen and the sero-fast preparation method of the antigenic routine of graininess (cell) all is the known technology of this area.After to any immunocompetent vertebrates injection allogenic material, all can produce polyclonal antibody.Ideal situation is to give antigen repeatedly to being used for producing sero-fast animal, till measuring the humoral response that has reached the suitableeest after the tentative bloodletting.Concerning immune purpose, any L/1C2 antigen positive material all is fit to.The anti-antiserum(antisera) that obtains can be measured as described in example 4 the antigenic reactive behavior of L/1C2.
Example 6
The detection of cancer and diagnosis
Utilize the avidin-vitamin H immunoperoxidase assay (Borowitz that revises, et al., American Journal of Clinical Pathology 79(3): 387-391,1983), illustrative antibody L/1C2 monoclonal antibody of the present invention is used as diagnostic tool.
Freezing tissue block is cut into size for 4-6 μ m, is added on the slide glass of gelatin bag quilt, air-dry after, with acetone fixed 5 minutes.After 5% horse serum pre-incubation, with tissue block successively with L/1C2 antibody, the anti-mouse Ig(Vectorlabs of biotinylated horse) and avidin and the horseradish peroxide mixture of crossing the thing enzyme be incubated, between these incubation step, (pH7.2-7.4) wash with the phosphate buffered saline(PBS) (PBS) that contains 1% bovine serum albumin(BSA).With 3,3 '-the diaminobenzidine colour developing, with the tissue block haematoxylin redyeing.
Example 7
Detection moiety combines with antibody of the present invention
Example 6 described methods are easy to by L/1C2 antibody biotinylation is made amendment, thereby have saved the needs to second antibody (the anti-mouse Ig of biotinylated horse).Utilize A protein chromatographic method purifying L/1C2 antibody, utilize dialysis or Sephadex G25
(Sigma) desalination, and make antibody enter 0.1M NaHCO
3, use 0.1M NaHCO
3Transfer pH to 8.5-8.6.The G25 post for preparing can be from Pharmacia chemical company as PD10
Post has been bought.With L/1C2 antibody at 0.1M NaHCO
3(pH8.5-8.6) concentration in is adjusted to 1mg/ml.With N-hydroxy-succinamide base vitamin H (Pierce Chemical Co., P.O.Box 117, Rockford Illinois61105) is dissolved among the DMSO fast, making its concentration is 1mg/ml, adds the L/1C2 antibody of 120 μ l/mg and mixes rapidly.Reaction mixture was placed under room temperature 4 hours.Bonded antibody can utilize dialysis method or make it pass through Sephadex G25
And itself and unreacted N-hydroxy-succinamide base vitamin H are separated.After the avidin binding substances reaction of biotinylated L/1C2 antibody and fluorescence half point, radioactive substance or enzyme, just help detecting.The use of avidin-vitamin H detection architecture is used widely in this area, and above-mentioned avidin binding substances can have been bought from many producers.
Can utilize the described method of document to make fluorescent marker (the Selected Methods in Cellular Immunology that directly links to each other with antibody of the present invention, P 292-302,1980, Mishell and Shiigi, ed., Freeman and Company, San Francisco), this document is classified this paper reference as.
Example 8
Isostructural mensuration
The homotype of the L/1C2 antibody that is produced by L/1C2 hybridoma (ATCC #B9862) is measured as IgG3.What use when carrying out this mensuration is to derive from the inferior homotype classification agent box of muroid mono-clonal of HyClone Laboratories and the specification sheets that the said firm provided.
Example 9
The inhibition of tumor cell extracorporeal growth
With 1 * 10
4Individual target cell is placed in each apertures of 96 hole tissue culture plate (Costar), under 37 ℃, is containing 5%CO
2Moist incubator in cultivated 16 hours, substratum is leucine auxotrophy type substratum (do not have leucic DME and add 13 μ g/ml L-leucines, 29.2 μ g/ml L-glutaminate, 50 μ g/ml gentamicins and 10% foetal calf serum of dialysing).All tissue culture reagent all derive from Gibco Laboratories.The aseptic then substratum of removing is added in the antibody diluent in the 200 μ l leucine auxotrophy type substratum.Flat board was cultivated 48 hours, removed substratum this moment and use instead and be added with
3The leucine auxotrophy type substratum (Gibco) of H-leucine (NEN) makes every aperture reach 4 μ Ci.Flat board put back in the incubator again cultivated 24 hours.Utilizing automated cell to collect instrument and liquid dodges technical measurement and mixes radioactivity in the macromole.
Example 10
Utilize the ultraviolet microtechnique to estimate L/1C2
Internalization in the T222 tumour cell
Handle the T222 cell with pancreatin/EDTA(Gibco), till observing the disengaging of cell and culturing bottle, from tissue culture flasks, collect the T222 cell with this method.With cell Hanks balanced salt solution (HBSS(Gibco)) washed twice.With 1 * 10
6Individual cell moves into 12 * 75mm centrifuge tube, centrifugal settling, sucking-off HBSS.Every pipe adds 50 μ l L/1C2(5 μ g/ml), be incubated 30 minutes on ice.After the insulation, cell is washed twice with HBSS, remove all unconjugated L/1C2, last washing back sucking-off HBSS.Add the fluorescein-labeled sheep F(ab ' of 50 μ l)
2Anti-mouse IgG(Tago), mixed content thing and be placed on and be incubated 30 minutes on ice then.Then cell is washed twice with HBSS, remove all unreacted fluorescein-labelled sheep F(ab ')
2Anti-mouse IgG thinks the zero point of minute constantly with this.
Example 11
Utilize
125L/1C2 is estimated in the I-L/1C2 endocytosis
Internalization in the T222 tumour cell
Utilize Iodobeads(Pierce Chemical Co., Rockford IL), use by the manufacturer's recommended method
125The L/1C2 of I mark purifying.Estimating
125During the internalization of I-L/1C2, think that cpm in the cell precipitation is internalization
125I-L/1C2(Malzku et al., Cancer Research 43: 3848,1986).Briefly be exactly to make 1 * 10
6Individual target cell and 200, the antibody of 000cpm mark reacted 90 minutes down at 0 ℃ or 37 ℃, washed 4 times, was suspended in the 0.5ml glycine buffer (0.05M glycine-HCl, pH2.8 and 0.1M NaCl), with the immunoglobulin (Ig) of dissociated cell surface bonding.After centrifugal, measure the radioactivity in supernatant liquor or the cell precipitation.Think that the radiocounting in the supernatant liquor is the cell surface bonded
125I-L/1C2.
Example 12
For estimating the nude mice that anti-tumor activity carries out
The xenotransplantation screening
To derive from Charles River Breeding Laboratories(Boston, outbreeding nude mice circle MA) gives to not timing sterilized water and food in Bechtop.Get the nude mice at about 2 monthly ages, coerce 1 * 10 among the subcutaneous vaccination 0.2ml PBS on the right side
7Individual tumour cell.The compound of preparation antitumor effectiveness to be evaluated notes reducing the wound and the infection of experimental animal injection site for intravenous administration as far as possible when using in aseptic PBS.
Determine curative effect by the size of checking tumour.On both direction, measure tumour afterwards, calculated size with following formula: length x width
2/ 2.Calculate tumor growth restraining effect with respect to the control animals tumor growth in vivo that only gives PBS.All treatment group and control group are one group with 10 mouse all.
Nude mice is preferred rodent to this test system, but various research is also carried out immunosuppression with radiation or immunosuppressor to other rodents, thus the immunoreactivity of blocking-up host animal.The T222 cell is preferred human tumor cell line to the bare mouse different species transplantation model, but the human tumor cell line that has only those expression L/1C2 antigens also can repeatedly set up tumour in nude mouse just might also be preferred.
Example 13
L/1C2-4-deacetylation vincaleucoblastine-3-
The preparation of carboxyl hydrazides
The oxidation of A.L/1C2 antibody
With L/1C2 antibody at 0.1M sodium acetate buffer pH5.6(acetate buffer) in thoroughly dialysis, being diluted to concentration is 15.7ml for the 10mg/ml(cumulative volume), make its in ice-water bath balance to about 0 ℃.Add 535mg solid (partially) sodium periodate, stirred reaction mixture dissolves up to sodium periodate, because of vigorous stirring can be damaged antibody, so will avoid gently.With reaction mixture stir about 21 minutes in ice bath, build with aluminium foil simultaneously and prevent the illumination mixture.Be incubated after 21 minutes, add 1 μ l 12.5M ethylene glycol (aqueous solution) and stop oxidizing reaction.By the centrifugal aggregation of from reaction mixture, removing, with clear soln at Sephadex G-25
Gel-filtration column (44g Sephadex-G25
, 2.5 * 50cm post) on carry out chromatography, this post is used the acetate buffer balance in advance.Collection is effusive first peak from post, is diluted to 2.77mg/ml with acetate buffer.Utilize 280nm determination of uv absorption protein concentration, the optical extinction coefficient of supposing the 1mg/ml antibody-solutions is 1.43.
B. oxidized form L/1C2 antibody and 4-deacetylation vincaleucoblastine-3-carboxyl hydrazides vitriol combines
To the front steps A obtain in about 0 °-2 ℃ oxidized form L/1C2 antibody (solution of 2.77mg/ml in acetate buffer that stir down; cumulative volume is 55ml) in; add 238mg4-deacetylation vincaleucoblastine-3-carboxyl hydrazides vitriol (pressing the preparation of EPO bulletin 247,792 disclosed methods).After the dissolving of 4-deacetylation vincaleucoblastine-3-carboxyl hydrazides vitriol, this mixture wrapped in prevent illumination in the aluminium foil, and move in cold house's (4 ℃), stir about is 16 hours in the cold house.Then by the centrifugal clarification reaction mixture, and at Sephadex G-25 gel-filtration column (67g Sephadex G-25
, 2.5 * 75cm post) on carry out chromatography, this post is used the PBS balance in advance.Collect each component, collect first peak and utilize dual-wavelength ultraviolet spectrophotometry to measure albumen and drug level at 280nm and 270nm place.Behind 0.2 μ m filtration sterilization (Millipore), store immune conjugate down in 4 ℃.
Example 14
L/1C2 propionyl-3-aminocarboxyl-4-
The preparation of phenyl aldehyde-methotrexate hydrazides
A. the preparation of methotrexate-γ-hydrazides
In the 100ml flask, add the 1.61g(10 mmole) L-L-glutamic acid 5-methyl ester.To wherein adding the 60ml ra-butyl acetate, stir this mixture to two kind of compound thorough mixing.Then under continuously stirring to wherein dripping the 1.58g(11 mmole) 70% perchloric acid.This mixture was stirred two days under nitrogen, divide three extractions with 100ml0.5N hydrochloric acid then.Combining water layer is also used the neutralization of 30g sodium bicarbonate.This neutral solution merges organic layer and uses the salt water washing with three parts of ether (cumulative volume is 150ml) extraction.Organic solution dried over sodium sulfate after will washing then, vacuum-evaporation under the room temperature obtains the 1.18g(5.4 mmole) clear thorough oily matter, be accredited as L-L-glutamic acid 5-methyl isophthalic acid-tertiary butyl diester.
To through adding the 0.92ml(6.6 mmole in the 100ml flask of oven drying) triethylamine, 1ml(6.6 mmole) dimethyl formamide that under vacuum, newly steams of diethyl phosphorocyanidate and 49ml with barium oxide.In the solution that stirs, add the 506mg(1.3 mmole) 2,4-diamino-6-(N-methyl-N-(4-carboxyl phenyl) amino) the pteridine trihydrate.Followed when this intermediate and to have stirred and after the dissolving,, added the 0.2ml(1.4 mmole that is dissolved in the 1ml dimethyl formamide then this mixture heating up to 80 ℃) the L-glutamic acid diester for preparing above of triethylamine and 342mg.This mixture was stirred 2 hours down in 80 ℃, then cooling and solvent removed in vacuo.Residue is dissolved in the 300ml chloroform, washs with 5% sodium hydrogen carbonate solution.Use the chloroform extraction water layer, merge organic layer, use dried over sodium sulfate, vacuum concentration gets the 1.35g orange, with its chromatography on 150g silica gel, with the eluant solution of 10% methyl alcohol in chloroform.Merge the fraction that contains product and also concentrate, get 645mg methotrexate diester, γ-carboxyl wherein is the methyl ester form, and α-carboxyl is the tertiary butyl ester form.
With above-mentioned intermediate and the same intermediate merging of another batch, total amount is the 697mg(1.3 mmole).It is dissolved in the 12ml methyl alcohol.To wherein adding the 170mg(5.3 mmole) anhydrous hydrazine, stir this mixture 6 days under room temperature and nitrogen atmosphere.Solvent removed in vacuo is carried out chromatography with residue on 150g silica gel then, and the chloroformic solution wash-out with 15% methyl alcohol obtains the 470mg(0.9 mmole) methotrexate-α-tert-butyl ester-γ-hydrazides.
Above-mentioned intermediate is dissolved in the 120ml 1N hydrochloric acid, heated 50 minutes down in 55 ℃.Vacuum concentration becomes solid then, residue is dissolved in the ammonium acetate of 80ml 0.01M pH8.This solution was stored 3 days down in 4 ℃, then at 300ml Sepharose Fast Flow Q(Pharmacia, Inc., Piscataway, NJ) carry out chromatography on, carry out wash-out in the gradient mode with the ammonium acetate (buffer B) of same damping fluid of top exhausted just (buffer A) and 1.0M pH8.What be incorporated in that wash-out goes out in 100% buffer B contains the product fraction, and frost drying is to remove the small amount of acetic acid ammonium repeatedly.Output is the 326mg(0.7 mmole) methotrexate-γ-hydrazides.
B. propionic acid N-succinimido ester linking group-3-(4-formylphenyl carbonylamino)
In the 250ml flask, add the 3g(20 mmole in the 100ml diox) 4-carboxyl benzaldehyde and 2.3g(20 mmole) N-hydroxy-succinamide.This mixture was stirred 5-10 minute, adds the 4.1g(20 mmole then) dicyclohexylcarbodiimide.This mixture was at room temperature stirred 1 hour, filter then.Vacuum-evaporation filtrate gets the 9.4g white solid, with the hot Virahol recrystallization of 25ml.Develop this intermediate product with Virahol, the 2.1g(8.5 mmole) the N-succinimido ester of required 4-carboxyl benzaldehyde.
Preparation is several intermediate products in addition, altogether the 10g(40 mmole) N-succinimido ester is added to the 3.6g(40 mmole) in the solution of Beta-alanine in 40ml 1N sodium hydroxide and about 100ml water.This mixture was stirred 1.5 hours, keep pH simultaneously more than 8.Then this mixture is filtered, filtrate uses the 2N hcl acidifying to pH1.9.With the ethyl acetate extraction of 150ml altogether 3 times, merge organic layer and use the salt water washing.Use the dried over sodium sulfate organic layer then, vacuum-evaporation gets 4.6g (21 mmole) white solid, i.e. 3-(4-formylphenyl carbonylamino) propionic acid.
In a little flask, add the 100mg(0.45 mmole) above-mentioned intermediate, 103mg(0.5 mmole) dicyclohexylcarbodiimide, 57.5mg(0.5 mmole) N-hydroxy-succinamide and 10ml diox, stir this mixture under room temperature and nitrogen.Utilize thin layer chromatography observing response process, add the 75mg(0.36 mmole again) dicyclohexylcarbodiimide and 45mg(0.4 mmole) N-hydroxy-succinamide.After 4 hours, filter reaction mixture, vacuum-evaporation filtrate is to solid.Obtain the impure product of about 200mg, on 30g silica gel, carry out chromatography, with the dichloromethane solution wash-out of 5% Virahol.Merge the fraction contain product, evaporate the 81mg(0.25 mmole) the required intermediate of impure slightly form.
C. make L/1C2 antibody and linking group-propionyl-3-aminocarboxyl-4-phenyl aldehyde coupling
Under room temperature, in the 100ml flask, add the 1026mg(6.8 micromole) solution of antibody L/1C2 in 76.1ml 0.34M borate buffer pH8.6, then add the 14.1mg(44 micromole) " preparation 3 " active ester solution in the 3.3ml acetonitrile of obtaining.This mixture was stirred under room temperature 1 hour.Then at 90g Sephadex G25(Pharmacia) on carry out chromatography, with the 0.1M sodium acetate wash-out of pH5.6.Measure each fraction by carrying out ultra-violet analysis, merge and contain the product fraction at 258nm and 280nm place, the 948mg(6.3 micromole) required product, its form is that concentration is the 111.5ml solution of 8.5mg/ml.The binding ratio of derived antibody is 4.8 moles of linking groups of every mole of antibody.
D. antibody L/1C2 propionyl-3-aminocarboxyl-4-phenyl aldehyde combines with methotrexate-γ-hydrazides
From the product of " preparation 4 ", get and contain the 472mg(3.1 micromole) part of the 55.6ml of derived antibody, in addition with 87ml 0.1M sodium acetate pH5.6 dilution.
With a 101mg(216 micromole) methotrexate-γ-hydrazides be dissolved in 7.2ml acetonitrile and the 21.6ml 1M potassium phosphate buffer.In this solution, add about 1ml 5N sodium hydroxide, then solution is added in the antibody-solutions.With glacial acetic acid reaction mixture is acidified to pH5.8, under room temperature, stirred 16 hours.Centrifugal then, supernatant liquor is carried out chromatography on two 90g Sephadex G25 chromatography columns, with physiological buffer salt water elution.
Collecting total amount by chromatography is the solution of 202.8ml, utilizes ultraviolet spectroscopy analysis, observes the spectrum at 280nm and 370nm place.Analysis revealed contains 0.018mg/ml methotrexate and 1.87mg/ml antibody in this solution.Therefore binding ratio counts 3.0 by mole number.Make the product solution concentration by at low temperatures physiological buffer salt solution being carried out the vacuum dialysis, with product solution of this example and the 212ml product solution merging that obtains by similar operations.Make volume reduce to 108ml by dialysis.
Example 15
Antibody L/1C2 propionyl-3-ammonia
Base carbonyl-4-phenyl aldehyde and methotrexate
The binding substances of-γ-hydrazides-change reaction conditions with
Generate the binding substances of various different binding ratio
The described association reactions operation of example 14 is repeated four times, reaction conditions and example 14 described conditions different only be following some.
A. methotrexate-γ-the hydrazides with a 0.67mg is dissolved in 67 μ l 0.1M sodium acetate pH5.6 and the 33 μ l acetonitriles, this solution and 1ml are contained the solution merging of 2.2mg example 14C intermediate partly in the 0.1M sodium acetate, this mixture was left standstill 11 hours.Then at 5.9g Biogel P6(Bio-Rad Laboratories, Richmond, CA 94804) on carry out chromatography, 5ml binding substances solution, wherein contain the required binding substances of 0.6mg, its binding ratio is every mole of antibody 3.2 moles of ammonia methopterin-As.
B. the solution that the methotrexate-γ-hydrazides solution and the 0.95ml of the used equivalent of example 15A part contained 0.42mg example 14C part intermediate was placed this mixture 11 hours.Carry out chromatography as described in example 15A part, get 4.75ml binding substances solution, wherein contain the 0.11mg binding substances, its binding ratio is every mole of L/1C2 antibody 5.4 moles of ammonia methopterin-As.
C. will be dissolved in the methotrexate-γ-hydrazides of a 0.7mg among 50 μ l acetonitriles and the 100 μ l 1M phosphoric acid buffer pH5.8, merge with the 2.2mg example 14C part intermediate that is dissolved among the 1ml 0.1M sodium acetate pH5.6.This mixture was placed 11 hours.Carry out chromatography then as described in example 15A part, get 5.3ml binding substances solution, wherein contain the 1.3mg binding substances, its binding ratio is every mole of antibody 3.3 moles of ammonia methopterin-As.
D. will be dissolved in the methotrexate-γ-hydrazides of a 0.7mg among 50 μ l acetonitriles and the 150 μ l 1M phosphoric acid buffer pH5.8, merge with the solution of 2.2mg example 14C part intermediate in 1ml 0.1M sodium acetate.This mixture was placed 14 hours, carried out chromatography then as described in example 15A part, get 5.3ml binding substances solution, wherein contain the 1.7mg binding substances, its binding ratio is every mole of L/1C2 antibody 3.3 moles of ammonia methopterin-As.
An individual system has been set up in the variation of above-mentioned association reaction operation, can control the degree of medicine and L/1C2 antibodies by this system.It is generally acknowledged that preferred binding ratio is the maximum number at obvious following the medicine that can connect of situation that descends of the antigen-binding matter that does not cause L/1C2 antibody.
Example 16
A. the preparation of antibody L/1C2 carbonyl-4-phenyl aldehyde intermediate
As described in example 14B part, prepare the 4-carboxyl benzaldehyde N-succinimido ester of a 0.31mg.It is dissolved in the 100 μ l dimethyl formamides, and it is added in the solution of 18.9mg antibody L/1C2 in 2.1ml 0.34M borate buffer pH8.6.Stirred this mixture 1.5 hours under room temperature, chromatography on 6g Biogel P6 is used 0.1M sodium acetate pH5.6 wash-out then.Obtain the solution of a 6.2ml, it is carried out ultra-violet analysis, observe the spectrum at 256nm and 280nm place.Analysis revealed obtains the 16.1mg derived antibody, and concentration is 2.6mg/ml, and its binding ratio is 5.2 moles of linking groups of every mole of antibody.
B. the preparation of the binding substances of antibody L/1C2 carbonyl-4-phenyl aldehyde and methotrexate-γ-hydrazides
Contain the 10.4mg(0.069 micromole with what 4ml derived from example 16A part) solution of derived antibody, with the 3.2mg(6.8 micromole) solution of methotrexate-γ-hydrazides in 250 μ l dimethyl formamides merges.This mixture was placed under room temperature 6 hours, be placed in the refrigerator then.Two days later, centrifugal this mixture is got supernatant liquor and carry out chromatography on Bio-Gel P6, with physiological buffer salt water elution, gets 8.5ml solution, it is carried out ultra-violet analysis, the spectrum at observation 280 and 370nm place.Analysis revealed, the binding ratio of this product are 6.4 moles of medicines of every mole of antibody, contain the 0.64mg/ml binding substances in this solution.
As described in example 9, estimate the ability that this product suppresses the T222 growth of tumour cell in the tissue culture.Find that this binding substances is that 0.035 μ g/ml(is in methotrexate content in concentration) time, cell growth produces 70% and suppresses.
Example 17
The preparation of L/1C2 Fab and binding substances thereof
A. antibody L/1C2F(ab ')
2Fragment
The F(ab ' for preparing antibody L/1C2 with the following method)
2Fragment: in the solution of 1.5gL/1C2 antibody in 270ml physiological buffer salt solution, add the pepsin solution that 2.4ml contains 12.6mg stomach en-/ml.This mixture was placed 2 hours 20 minutes down in 37 ℃, added the triethylamine stopped reaction then.The enriched product by chromatography on Sepharose Fast Flow post is used 0.15M sodium acetate wash-out then.Merge and contain F(ab ')
2Fraction, concentrate by dialysis, 100ml product solution, wherein contain the F(ab ' of 992mg antibody L/1C2)
2Fragment.
B. antibody L/1C2F(ab ')
2The preparation of fragment propionyl-3-aminocarboxyl-4-phenyl aldehyde intermediate
The L/1C2F(ab ' that this example 17A is partly prepared)
2Fragment carries out dialysis among the 0.34M borate buffer pH8.6, gets the 23mg(0.23 micromole) F(ab ')
2Fragment, its form are 3.8ml solution.With this solution and 0.44mg(1.4 micromole) 3-(4-formylphenyl carbonylamino) the solution merging of propionic acid N-succinimido ester in 102 μ l acetonitriles.This mixture was stirred under room temperature 1 hour, then this solution is carried out chromatography on the post of 11g Sephadex G25, with 0.1M sodium acetate pH5.6 wash-out.Merge and contain the product fraction, get the 19mg(0.19 micromole) the derived antibody fragment, its binding ratio is 2.8 moles of linking groups of every mole of antibody in 9.6ml solution.
C.L/1C2F(ab ')
2The preparation of the binding substances of fragment propionyl-3-aminocarboxyl-4-phenyl aldehyde and methotrexate-γ-hydrazides.
Contain the 8mg(0.08 micromole to what 2.7ml front B partly prepared) in the segmental solution of derived antibody, add 0.47ml 1M phosphoric acid buffer pH5.6.Add the 3.7mg(7.9 micromole who is dissolved in the minimum 0.1M sodium acetate to this solution) methotrexate-γ-hydrazides.PH with mixture recalls to 5.6 with dilute hydrochloric acid, stirs this mixture 17 hours under room temperature.Centrifugal then, get supernatant liquor and on the post of 11g Sephadex G25, carry out chromatography, with physiological buffer salt water elution.Ultra-violet analysis shows, has obtained the 5.8mg binding substances, and its form is the solution that contains the 0.95mg/ml binding substances, and its binding ratio is every mole of L/1C2 antibody 2.1 moles of ammonia methopterin-As.
As described in example 9, the test binding substances is to the activity of T222 tumour cell in tumor cell extracorporeal growth suppresses to measure, the discovery binding substances suppresses this cell when concentration is 0.0046 μ g/ml growth reaches 22%, suppresses growth 83%(concentration in methotrexate-γ-hydrazides content when concentration is 0.046 μ g/ml).
Claims (18)
1, a kind of tumor-associated glycoprotein antigen of substantially pure form, described antigen has following character: utilize SDS-PAGE to record molecular weight 110 under reductive condition, 000-140 is in 000 dalton's scope; Exist down in coming from head; People's squamous epithelium cancer cell surface of neck and pulmonary epithelial cells; The antibody mediated immunity precipitation that can be produced by hybridoma cell line L/1c2.
2, a kind of hybridoma cell line, its produce can with the antibody of the described glycoprotein antigen reaction of claim 1.
3, the hybridoma cell line of claim 2 is characterized in that, it is L/1C2.
4, a kind of can with the antigen reactive antibody of the described tumor-associated glycoprotein of claim 1.
5, a kind of antibody that produces by the hybridoma cell line of claim 2.
6, the antibody of claim 4 is characterized in that, it is a kind of chimeric antibody.
7, claim 4 or 5 antibody is characterized in that it is a kind of IgG antibody.
8, the antibody of claim 7 is characterized in that, it is a L/1C2 antibody.
9, a kind of molecule that contains an antigen binding domain of each antibody among the claim 4-8.
10, the Fab fragment of each antibody among claim 4-8.
11, the F(ab ' of each antibody among claim 4-8)
2Fragment.
12, whether have the antigenic method of L/1C2 in a kind of test sample, it comprises: (a) add a kind of antibody, the described tumor-associated glycoprotein antigen-reactive of this antibody capable and claim 1 in described sample; (b) reactive behavior of described antibody of mensuration and described sample.
13, a kind of carcinostatic agent that is used for the treatment of the human body cancer, it contains each described antibody, molecule or fragment among the claim 4-11, they and a kind of radioelement, biotoxin or molten cancer drug coupling.
14, a kind of carcinostatic agent as claimed in claim 13 is characterized in that, biotoxin is dipteral insect toxin or ricin, and perhaps molten cancer drug is methotrexate, a kind of vinca alkaloids or adriamycin.
15, a kind ofly it is characterized in that as claim 13 or 14 described carcinostatic agents described antibody, molecule or fragment are the secretory product monoclonal antibody L/1C2 of ATCC#B9682.
16, a kind of treatment human body method for cancer, it comprise use to human body a kind of as each described carcinostatic agent among the claim 13-15.
17, a kind of preparation is as each described antibody, molecule or segmental method among the claim 5-11, and it comprises: (a) make claim 2 or 3 described hybridoma cell lines secrete a kind of monoclonal antibody; (b) the described monoclonal antibody of purifying; (c) prepare Fab or F(ab ' as need) fragment, can optionally cut described antibody purification with proteolytic ferment papoid or stomach en-.
18, a kind of preparation is as the method for each described carcinostatic agent among the claim 13-15, and it comprises: make a kind of as each described antibody, molecule or fragment and a kind of radioelement, biotoxin or molten cancer agent coupling among the claim 4-11.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US18465988A | 1988-04-22 | 1988-04-22 | |
| US184,659 | 1988-04-22 | ||
| US259,679 | 1988-10-19 | ||
| US07/259,679 US5171666A (en) | 1988-04-22 | 1988-10-19 | Monoclonal antibodies reactive with a cell-surface gylcoprotein expressed on human carcinomas |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1037925A true CN1037925A (en) | 1989-12-13 |
Family
ID=26880357
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN89102525A Granted CN1037925A (en) | 1988-04-22 | 1989-04-21 | Monoclonal antibody |
Country Status (11)
| Country | Link |
|---|---|
| US (1) | US5171666A (en) |
| EP (1) | EP0338846A3 (en) |
| JP (1) | JPH0235097A (en) |
| KR (1) | KR900015756A (en) |
| CN (1) | CN1037925A (en) |
| AU (1) | AU612824B2 (en) |
| DK (1) | DK191989A (en) |
| HU (1) | HUT52166A (en) |
| IL (1) | IL90017A0 (en) |
| NZ (1) | NZ228837A (en) |
| PT (1) | PT90323A (en) |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5134075A (en) * | 1989-02-17 | 1992-07-28 | Oncogen Limited Partnership | Monoclonal antibody to novel antigen associated with human tumors |
| US5171665A (en) * | 1989-04-17 | 1992-12-15 | Oncogen | Monoclonal antibody to novel antigen associated with human tumors |
| ZA914625B (en) * | 1990-06-22 | 1993-02-24 | Lilly Co Eli | In vivo targeting with bifunctional antibodies |
| CA2044421C (en) * | 1990-11-27 | 2005-06-21 | Thomas Hyatt Duffy | Squamous cell carcinoma-like immunoreactive antigen from human female urine |
| JPH06145196A (en) * | 1992-11-10 | 1994-05-24 | Tosoh Corp | Protein secreted by cancer cell |
| US5424402A (en) * | 1993-10-18 | 1995-06-13 | Board Of Trustees Of The University Of Kentucky | Non-destructive method for radiolabelling biomolecules by halogenation |
| US5482040A (en) * | 1994-04-14 | 1996-01-09 | The Ohio State University Research Foundation | Biostaging of adenocarcinomas utilizing radiolabeled tumor-associated glycoprotein antibodies |
| US6348194B1 (en) * | 1995-11-13 | 2002-02-19 | Ixsys Incorporated | Tumor specific internalizing antigens and methods for targeting therapeutic agents |
| US6406917B1 (en) | 1996-06-11 | 2002-06-18 | Advanced Research And Technology Institute | Methods and compositions for the use of apurinic/apyrimidinic endonucleases |
| WO1997047971A1 (en) | 1996-06-11 | 1997-12-18 | Advanced Research & Technology Institute | Methods and compositions for the use of apurinic/apyrimidinic endonucleases |
| AU2003220091B2 (en) * | 2002-03-08 | 2006-02-16 | Emory University | Novel curcuminoid-factor VIIa constructs as suppressors of tumor growth and angiogenesis |
| US20050069551A1 (en) * | 2002-03-08 | 2005-03-31 | Emory University | Cytotoxic compound-protein conjugates as suppressors of tumor growth and angiogenesis |
| AU2015224535B2 (en) * | 2011-06-21 | 2017-07-20 | Immunogen, Inc. | Novel maytansinoid derivatives with peptide linker and conjugates thereof |
| CA2836927A1 (en) * | 2011-06-21 | 2012-12-27 | Immunogen, Inc. | Novel maytansinoid derivatives with peptide linker and conjugates thereof |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4132769A (en) * | 1974-10-30 | 1979-01-02 | Osther Kurt B | Cancer antigen, cancer therapy, and cancer diagnosis |
| US4713352A (en) * | 1981-08-31 | 1987-12-15 | Sloan-Kettering Institute For Cancer Reseach | Monoclonal antibody panel for early diagnosis and therapy of renal carcinoma |
| US4816567A (en) * | 1983-04-08 | 1989-03-28 | Genentech, Inc. | Recombinant immunoglobin preparations |
| CA1320689C (en) * | 1984-03-30 | 1993-07-27 | Alain Schreiber | Monoclonal antibody specific for human basal cell surface antigen |
| US4708930A (en) * | 1984-11-09 | 1987-11-24 | Coulter Corporation | Monoclonal antibody to a human carcinoma tumor associated antigen |
| US5019497A (en) * | 1984-11-09 | 1991-05-28 | Lennart Olsson | Human squamous lung carcinoma cell specific antigens and antibodies |
| US4814275A (en) * | 1985-05-13 | 1989-03-21 | E.I. Dupont De Nemours And Company | Monoclonal hybridoma antibody specific for a human epithelial antigen |
| US4816402A (en) * | 1986-01-07 | 1989-03-28 | Northwestern University | Murine hybridoma and diagnostic antibody produced thereby |
| EP0232871A3 (en) * | 1986-02-07 | 1989-03-15 | Yoshitomi Pharmaceutical Industries, Ltd. | Human monoclonal antibody, hybridoma producing the same and its use |
| US4912031A (en) * | 1987-10-23 | 1990-03-27 | E. I. Du Pont De Nemours And Company | Method for detecting carcinomatous and precarcinomatous colo-rectal disease |
-
1988
- 1988-10-19 US US07/259,679 patent/US5171666A/en not_active Expired - Fee Related
-
1989
- 1989-04-18 IL IL90017A patent/IL90017A0/en unknown
- 1989-04-20 PT PT90323A patent/PT90323A/en not_active Application Discontinuation
- 1989-04-20 DK DK191989A patent/DK191989A/en unknown
- 1989-04-20 NZ NZ228837A patent/NZ228837A/en unknown
- 1989-04-21 KR KR1019890005264A patent/KR900015756A/en not_active Application Discontinuation
- 1989-04-21 EP EP89303978A patent/EP0338846A3/en not_active Ceased
- 1989-04-21 AU AU33306/89A patent/AU612824B2/en not_active Ceased
- 1989-04-21 JP JP1103321A patent/JPH0235097A/en active Pending
- 1989-04-21 CN CN89102525A patent/CN1037925A/en active Granted
- 1989-04-21 HU HU891940A patent/HUT52166A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| AU3330689A (en) | 1989-10-26 |
| US5171666A (en) | 1992-12-15 |
| AU612824B2 (en) | 1991-07-18 |
| NZ228837A (en) | 1991-08-27 |
| DK191989D0 (en) | 1989-04-20 |
| DK191989A (en) | 1989-10-23 |
| PT90323A (en) | 1989-11-10 |
| JPH0235097A (en) | 1990-02-05 |
| IL90017A0 (en) | 1989-12-15 |
| EP0338846A2 (en) | 1989-10-25 |
| HUT52166A (en) | 1990-06-28 |
| KR900015756A (en) | 1990-11-10 |
| EP0338846A3 (en) | 1990-03-28 |
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| C01 | Deemed withdrawal of patent application (patent law 1993) | ||
| WD01 | Invention patent application deemed withdrawn after publication |